ODCase (EC 4.1.1.23) plays a central role in the de novo synthesis of uridine-5′-O-monophosphate (UMP). UMP is a building block, synthesized de novo from aspartic acid, for the synthesis of other pyrimidine nucleotides such as uridine-5′-O-triphosphate (UTP), cytidine-5′-O-triphosphate (CTP), thymidine-5′-O-triphosphate (TMP) and 2′-deoxy-cytidine-5′-O-triphosphate (dCTP) (FIG. 1). Pyrimidine nucleotides are the building blocks for the synthesis of RNA and DNA, the essential molecules for cell replication and survival. Due to its important role in the cell's de novo nucleic acid synthesis, ODCase is present in bacteria, archea, parasites and in humans, i.e. almost every species except in viruses. This enzyme catalyzes the decarboxylation of orotidine monophosphate (OMP) to uridine monophosphate (UMP) (compounds 1 and 2 in the final step in FIG. 1). This enzyme is particularly interesting for enzymologists because it exhibits an extraordinary level of catalytic rate enhancement of over 17 orders of magnitude compared to the uncatalyzed decarboxylation reaction in water at neutral pH 7.0, 25° C.i,ii An uncatalyzed decarboxylation occurs of OMP takes about 78 million years, and the enzymic decarboxylation at a millisecond time scale. Thus, ODCase is one of the most proficient members of the enzymic world.ii,iii,iv,v 
Interestingly, decarboxylases found in Nature use either a cofactor or covalent intermediates during the catalysis of decarboxylation reactions.vi,vii For example, thiamin diphosphate-dependent, indole pyruvate decarboxylase (IPDC) uses thiamin as a cofactor and there are covalent intermediates formed with the cofactor during the decarboxylation process. ODCase is thought to be quite unusual in catalyzing decarboxylation with such proficiency without the help of any co-factors, metals, or covalent-intermediates.i,ii,iii One interesting difference when one looks at this enzyme across species is that in certain higher level organisms such as human or mouse, ODCase is a part of the bifunctional enzyme, UMP synthase.viii In pathogenic organisms such as bacteria, fungi and parasites, ODCase is a monofunctional enzyme.ix,x In all species, ODCase (whether monofunctional or bifunctional) is active as a dimeric unit.
In general, investigations targeting ODCase focused on malaria, cancer and few antiviral investigations. In the past two decades, several analogs of OMP were investigated extensively to understand the catalytic mechanism of ODCase.iii,xi Among these analogs, 6-aza-UMP (3) and 6-hydroxy-UMP (or BMP, 4), pyrazofurin, xanthosine-5′-monophosphate (XMP, 12) and 6-thiocarboxamido-UMP (13) are some of the potent inhibitors that were studied against ODCase (FIG. 2).xii,xiii,xiv However, the development of inhibitor candidates has been limited due to their toxicities and lack of specificity.xii There is also very limited or non-existent structure-activity relationship investigations and inhibitor design against ODCase. Thus, ODCase has not gained much traction in 1980s and 1990s as a drug target.
Aside from its obvious pharmacological interest, ODCase has been a favorite enzyme for biochemists and structural biologists due to its unusual catalytic properties. A number of mechanisms were proposed prior to and after the availability of X-ray crystal structures for several ODCases in 2000.xv,xvi,xvii,xviii Although ideas of covalent catalysis were discussed, none of the mechanisms presented included a covalent species formation as a key step during the decarboxylation by ODCase. An analysis of the catalytic site of ODCase from Methanobacterium thermoautotrophicum (Mt) revealed two aspartate residues (Asp70 and Asp75B, the latter contributed by the second subunit of the dimeric ODCase) and two lysine residues (Lys42 and Lys72) that are held via a strong network of hydrogen bonds (FIG. 3). Analyses of several co-crystal structures of ODCase with a variety of ligands confirm that these residues are held tightly in their respective positions in the active site and there is less than 0.5 Å movement in the positions of the side chains of these residues. Existing evidence does not support any active site residue forming a covalent bond either to the substrate during catalysis or to any known inhibitor.iii,xvi,xix,xx,xxi The above four residues are proposed to exert strong steric and electrostatic stress onto the C-6 carboxylate group of OMP and eliminate the carboxyl group.xvi 
The x-ray crystal structures of ODCase from ten different species are known today. In 2000, four x-ray crystal structures of ODCase brought insights into the catalytic mechanism of this enzyme. Based on the structure of S. cerevisiae ODCase complexed with the transition-state analogue BMP (4), a transition-state stabilization mechanism of OMP decarboxylation was proposed.xviii A similar proposal was also suggested by Appleby et al. based on the crystal structure of ODCase (Bacillus subtilis) complexed with the product, UMP.xvii These authors suggested that the decarboxylation reaction proceeds via an electrophilic substitution in which C-6 is protonated by Lys62 as the carbon dioxide molecule is released.xvii The structure of the ODCase enzyme from E. coli co-crystallized with BMP was the basis of the proposal submitted by Harris et al.xxii Based on the proximity of the carboxylate moiety on OMP (1) and Asp71 residue in the active site of ODCase, it was proposed that OMP decarboxylation depends on the existence of a shared proton between Asp71 and the carboxyl group of the substrate.xxii A similar mechanism involving electrostatic repulsion was put forward by Wu et al.xvi This mechanism of OMP decarboxylation is based on the principles of the Circe effect described by Jencks in 1975.xxiii The Circe effect states that only the reactive group of the substrate needs to be destabilized. The strong interaction between the unreactive part of the substrate and the enzyme active site provides the energy to directly destabilize the reactive group of the substrate.xxiii The electrostatic repulsion mechanism points to the active site aspartate residue. In four different species the location and function of this residue is highly conserved. The catalytic residues, Asp70 and Lys72 are located near the reaction center C-6 of the pyrimidine ring of the substrate OMP and Asp70 (M. thermoautotrophicum) was postulated to provide the electrostatic destabilization of the enzyme-substrate complex. Lys72 in the active site furnishes the proton to neutralize the carbanion developed after the departure of the carboxylate.xvi Despite several x-ray structures and in-depth enzymology in the past two decades, ODCase continues to challenge biochemists with still-unresolved mechanism and new twists (vide infra).
In the active site of ODCase, the monophosphate group of OMP is proposed to bind first and this group contributes the largest energy required for the binding of the substrate to ODCase.xxiv The removal of phosphate from the molecule of substrate resulted in a significantly lower catalytic efficiency measured as the second-order rate constant (kcat/KM) for the catalysis of substrate to product.xxiv In an interesting experiment, the binding of phosphite dianion (HPO32−) to ODCase (from S. cerevisiae) resulted in an 80,000 fold increase in the second order rate constant for the decarboxylation of the truncated substrate lacking a phosphate moiety.xxv Thus, the phosphate group is an important component for ODCase binding. Thus, in order for nucleoside drugs (correct terminology is prodrugs) to be active against ODCase in vivo, the nucleoside compound has to be converted into its monophosphate form inside the cell by any nucleoside kinase and then inhibit ODCase (whether in a pathogen or human cell). This is very similar to other nucleoside drugs such as AZT, 3TC, gemcitabine among several nucleoside drugs that are clinically used, thus there is a good possibility for the “nucleoside forms” of ODCase inhibitors to function as drugs.
If one carefully analyzes the biochemical and pharmacological basis, ODCase is one of most fascinating enzymes as a drug target. For example, Plasmodia species such as P. falciparum and P. vivax are dependent on their own de novo synthesis of pyrimidine nucleotides due to the absence of the salvage pathway in these parasites.xxvi In human, however, pyrimidine nucleotides are synthesized via both the de novo and salvage pathways.xxvii Thus, inhibition of plasmodial ODCase has been proposed as a strategy to design compounds directed against malaria and limited number of orotate analogs were investigated as potential drugs against the malarial parasite.xii,xxviii,xxix Most of these compounds are very polar with poor pharmacokinetics problems.
There remains a need for new inhibitors of ODCase as therapeutic agents, for example, for the prevention and treatment of malaria.